Progress 04/01/07 to 12/31/11
Outputs
OUTPUTS: My laboratory studies the metabolic diversity of microorganisms living at high temperatures(above 85C)to understand their ecology and explore the applications of novel metabolic processes and enzymes. We have developed novel approaches to isolate hyperthermophilic cellulolytic microorganisms from hydrothermal vent samples using pebbled-milled cellulose as sole electron donor and using Fe(III) oxides as electron acceptor for growth. With this approach, we have isolated an Fe(III)-reducing hyperthermophile that continues to grow in successive transfers at 100C in an anaerobic medium with cellulose and Fe(III) oxide. This novel isolate can only use Fe(III) oxide as electron acceptor, strongly indicating that Fe(III) reduction coupled to organic matter degradation may be a novel metabolic process in hot ecosystems that can be harnessed for the isolation of novel cellulolytic hyperthermophilic strains. Interestingly, ethanol was the main product of fermentation.In this proposal, we will (i) further characterize the metabolism and biochemistry of this novel organism, (ii) screen our collection of novel hyperthermophilic isolates for growth on cellulose and for cellulase production, and (iii) recover other cellulolytic hyperthermophiles from similar hot environments. PARTICIPANTS: Krista Cosert, Caitlin Miller, Lauryn Prezluski, Jayme Olsen, Andrew Murray, Josh Cambridge, Sadhana Lal and Kazem Kashefi TARGET AUDIENCES: This research has profound implication in multiple scientific disciplines such as Microbiology, geology, biochemistry, astrobiology and geomicrobiology. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The use of Fe(III) as an electron acceptor for the characterization of known hyperthermophiles or for the enrichment and isolation of novel Fe(III) reducers at high temperatures, lead to discovery of unsuspected metabolic processes. As a result we have greatly increased the known metabolic capabilities of hyperthermophilic microorganisms. For example, as a part of a study of the microbial diversity of extreme environments, primary oil recovery samples were collected from an oil reservoir in Lost Hills, California. Crude oil samples collected from this reservoir were used to inoculate enrichment culture media containing a variety of electron acceptors paired with organic or inorganic electron donors. A pure culture was recovered on solidified medium under sulfate-reducing conditions and was designated strain Delta due to its triangular shape (about 0.7-1.0 micrometer wide). A graduate student, Krista Cosert, has been trained during her rotation while studying degradation of crude oil by this organism. During last year, we have also been studying novel hydrogenases from Fe(III) reducing hyperthermophiles. Hydrogenases (H2ases) are metalloproteins containing [NiFe]- or [FeFe]-active centers that catalyze the reversible reduction of molecular hydrogen (H2). The possibility of using hydrogenases for the production of hydrogen has attracted great interest. Research to date has identified hydrogenases in several prokaryotes but only a few are able to preferentially catalyze the H2 evolution reaction. Considering the vast diversity of microorganisms, both bacteria and archaea, that live in H2-rich environments, we sought to explore the hydrogenases in novel microbes with high H2 production potential. Biological H2 production by anaerobic hyperthermophiles, in particular, is attractive due to their potential to utilize wide range of substrates for biohydrogen production. In this study, we developed PCR-based methods and identified [NiFe]- and [FeFe]-hydrogenase sequences among novel Fe(III)-reducing hyperthermophiles such as the bacteria Geothermobacterium ferrireducens, and the archaeon Geoglobus ahangari. In addition, efforts have been made to identify [FeFe]-only among mesophilic bacteria to explore their potential for biohydrogen production. Characterization of the biochemical properties of these hydrogenases both in in vitro and in vivo assays thus offers the opportunity to understand and manipulate the mechanisms for H2 evolution.Parallel to our research efforts, we have devoted a great deal of the project to educational activities such as training undergraduate and graduate students. As a result, five undergraduate students, Caitlin Miller, Lauryn Prezluski, Jayme Olsen, Andrew Murray,and Josh Cambridge have been trained and are currently doing independent research.
Publications
- Kashefi, K. 2011. Hyperthermophiles: Metabolic Diversity and Biotechnological Applications. In Extremophiles: Microbiology and Biotechnology. Antinori, R., ed. Horizon Press.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Key to optimizing the enzymatic hydrolysis of lignocellulosic substrates is the identification of thermostable enzymes that perform optimally at high temperatures, which will increase the efficiency of the enzymatic step while keeping the cost at a minimum. A major limitation in the discovery of thermophilic biomass-degrading enzymes is the limited number of thermophilic strains available in pure culture and the production of cellulases by these strains is rare. Recently, new microorganisms capable of growth at even higher temperatures (above 85oC) (hyperthermophiles) have been isolated that show promise for industrial applications requiring high temperatures. These organisms inhabit hydrothermal vents and terrestrial hot springs, where lignocellulosic substrates are abundant, suggesting they have the ability to hydrolyze and ferment lignocellulosic substrates. My laboratory studies the metabolic diversity of microorganisms living at high (> 85C) temperatures to understand their ecology and explore the applications of novel metabolic processes and enzymes. We have developed novel approaches to isolate hyperthermophilic cellulolytic microorganisms from hydrothermal vent samples using pebbled-milled cellulose as sole electron donor and using Fe(III) oxides as electron acceptor for growth. With this approach, we have isolated an Fe(III)-reducing hyperthermophile that continues to grow in successive transfers at 100C in an anaerobic medium with cellulose as electron donor and Fe(III) oxide as sole electron acceptor. Ethanol was the main product of fermentation. We are characterizing the metabolism and biochemistry of this novel organism. In addition we are screening our collection of novel hyperthermophilic isolates for growth on cellulose and for ethanol production, as well as recover other cellulolytic hyperthermophiles from similar hot environments. Aiming at discovering new enzymes for the bioconversion of lignocellulose by characterizing potentially novel metabolic pathways that can be engineered in bacterial and yeast strains to remove fermentation byproducts and increase ethanol production rates. PARTICIPANTS: Lauryn Przeslawski, Caitlin Miller and Molly Griffin (undergraduate students) Gemma Reguera (collaborator) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: We have extended studies on the metabolic diversity of hyperthermophiles to cellulose degraders for enzyme discovery.
Impacts
Parallel to our research efforts mentioned above, we have devoted a great deal of the project to educational activities aimed at training undergraduate and graduate students. As a result, three female undergraduate students, Lauryn Przeslawski, Caitlin Miller and Molly Griffin have been trained and are currently doing independent research with. Lauryn is studying toxic and heavy metal reduction by several of our novel hyperthermophilic isolates. This has importance for the design of novel enrichment and isolation procedures. Furthermore, it has possible significance for the evolution of Fe(III) reduction because the high conservation of the capacity for Fe(III) reduction among hyperthermophiles suggests that Fe(III) reduction was an early form of respiration. Caitlin and Molly are helping with characterization of novel isolates. We have also provided materials for numerous writers of textbooks that are including our research in their books and worked with various media sources to help them publicize this research.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: A phylogenetically diverse group of microorganisms share the ability to conserve energy from the reduction of metals such as Fe(III) and greatly contribute to the geochemistry of modern environments, both at moderate and high temperatures. Because of their unusual metabolism Fe(III)-reducing microorganisms can play an important role in the bioremediation of subsurface environments contaminated with organic or metal contaminants. Some Fe(III) reducers may use molecular hydrogen as well as organic acids such as acetate as electron donors for the reduction of Fe(III) and thus also contribute greatly to the degradation of organic matter in their natural environments. PARTICIPANTS: Sadhana Lal, Ping Zhang, Caitlin Miller, Molly Griffin and Kazem Kashefi TARGET AUDIENCES: This research has profound implication in multiple scientific disciplines such as Microbiology, geology, biochemistry, astrobiology and geomicrobiology. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Research during last year concentrated on the isolation and characterization of hyperthermophilic microorganisms from geographically distant hydrothermal systems and the use of Fe(III) as an electron acceptor for the isolation and characterization of novel forms of microbial respiration and metabolisms in modern and ancient hot environments. As a result we have greatly increased the known metabolic capabilities of hyperthermophilic microorganisms. For example, primary oil recovery samples were collected from an oil reservoir in Long Beach, California, and enriched with various pairs of electron donors and acceptors. A pure culture was recovered on solidified medium under sulfate-reducing conditions and was designated strain Δ due to its triangular shape (about 0.7-1.0 μm wide). This strain is novel because it is the first hyperthermophile capable of growth both on Fe(III) and sulfate and as such, probably represents the evolutionary "missing link" between the Fe(III)-reducers in the genera Ferroglobus and Geoglobus and sulfate-reducing Archaeoglobus spesies. During last year, we have also screened Fe(III) reducing hyperthermophiles for novel hydrogenases with potential for H2 production. Biological H2 production by anaerobic hyperthermophiles, in particular, is attractive due to their potential to utilize wide range of substrates for biohydrogen production and superior performance under environmental stresses. Hydrogenases (H2ases) are metalloproteins containing [NiFe]- or [FeFe]-active centers that catalyze the reversible reduction of molecular H2. Research to date has identified hydrogenases in several prokaryotes but only a few are able to preferentially catalyze the H2 evolution reaction. In this study, we developed PCR-based methods and identified [NiFe]- and [FeFe]-hydrogenase sequences among novel Fe(III)-reducing hyperthermophiles. In addition, efforts have been made to identify [FeFe]-only among mesophilic bacteria to explore their potential for H2 production. Characterization of the biochemical properties of these hydrogenases both in in vitro and in vivo assays thus offers the opportunity to understand and manipulate the mechanisms for H2 evolution. Parallel to our research efforts, we have devoted a great deal of the project to educational activities such as training undergraduate and graduate students who are currently doing independent research with us since last summer.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Our project's major objective is the isolation and characterization of novel hyperthermophilic Fe(III) reducers as a means to gain insight into the processes of microbial respiration taken place in modern and ancient hydrothermal environments. Modern hot environments such as those found in hydrothermal vents are of great interest to the scientific community because they resemble those ancient environments where life first arose on Earth and also share many characteristics with environments in other planets where life may have actually existed. Evidence to date strongly indicates that Fe(III) respiration may have been one of the first, if not the first, forms of respiration in a hot, early Earth. The abundance of Fe(III) minerals in many modern and ancient hot environments suggests that studies of Fe(III)-reducing hyperthermophilic microorganisms are likely to be instrumental in understanding how life originated and evolved at high temperatures. The isolation and characterization of novel hyperthermophilic, Fe(III)-reducing microorganisms has greatly increased our understanding on how microbes can live and thrive in such inhospitable environments. The study of these amazing microbes cues us into how life might have arisen on Earth and has implications in our search for traces of life in other planets. PARTICIPANTS: Jordan Elizabeth Habitz, Robert D. Burns and Dr. Ping Zhang. TARGET AUDIENCES: This research has profound implications in multiple scientific disciplines such as Microbiology, Geology, Biochemistry and Astrobiology. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Research during the last years was concentrated on the isolation and characterization of hyperthermophilic microorganisms from geographically distant hydrothermal systems and the use of Fe(III) as an electron acceptor for the isolation and characterization of novel forms of microbial respiration and metabolisms in modern and ancient hot environments. We also made significant progress in understanding additional environmentally relevant forms of Fe(III) that can be used for recovering novel hyperthermophiles in culture and in the process also made findings that have important geological implications. Our previous studies have been restricted to the use of poorly crystalline Fe(III) oxides as the Fe (III) source for enrichment and isolation. Although this has yielded numerous novel isolates, in many hot, sedimentary environments the Fe(III) may be in different forms. One of the most important source of Fe(III) in many sediments is the structural Fe(III) found in clays. Therfore a wide diversity of thermophilic and hrperthermophilic Archaea and Bacteria from marine and freshwater environments that are known to reduce poorly crystalline Fe(III) oxides were tested for their ability to grow with structural Fe(III) in smectite as the electron acceptor. Two out of ten organisms tested, Geoglobus ahangari and Geothermobacterium ferrireducens, were not able to conserve energy to support growth by reduction of structural Fe(III) in smectite. Interestingly, both these organisms were isolated on Fe(III) oxide and are not known to use any other electron acceptor. The other eight organisms tested were able to grow on smectite, reducing the structural Fe(III). Two organisms, Geothermobacter ehrlichii strain SS015 and archael strain 140, which produced copious amounts of an exopolysaccharide, dissolved a substantial portion (69 and 88% respectively) of Fe(II) that was produced from the smectite. It has previously been suggested that mesophilic Fe(III)-reducing microorganisms play a role in the transformation of smectite to illite. However no smectite-to-illite transformation as the result of Fe(III) reduction by hyperthermophiles was detected with X-ray diffraction analysis. More detail analysis of reduced by the hyperthermophile, strain 140, reveled that reduction of Fe(III) in the smectite was accompanied by increase interlayer and octahedral charges and some incorporation of potassium into the structure. These results suggest that hyperthermophilic Fe(III)-reducing microorganisms differ in their ability to reduce and solubilize the structural Fe(III) of phyllosilicates and that clays may provide an alternative Fe(III) source, which if used in initial enrichment and isolation of samples from hot environments, is likely to lead to novel isolates. These results have important geological as well as microbiological implications. Parallel to our research efforts, we have devoted a great deal of the project aimed at training undergraduate and graduate students as well as postdoctoral research associates.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Research during this first year has concentrated on studying whether the reduction of other Fe(III)-containing minerals can occurred at hot temperatures. Recent studies have suggested that the structural Fe(III) within phyllosilicate minerals, including smectite and illite, is an important electron acceptor for Fe(III)-reducing microorganisms in sedimentary environments at moderate temperatures. The reduction of structural Fe(III) by thermophiles, however, has not previously been described. A wide range of thermophilic and hyperthermophilic Archaea and Bacteria from marine and freshwater environments that are known to reduce poorly crystalline Fe(III) oxides were tested for their ability to reduce structural (octahedrally coordinated) Fe(III) in smectite (SWa-1) as the sole electron acceptor. Eight out of the ten organisms tested were able to conserve energy to support growth by reduction of Fe(III) in SWa-1 d and reduced 6.3 to 15.1 % of the Fe(III). This is 20-50% less
than the reported amounts of Fe(III) reduced in the same smectite (SWa-1), by mesophilic Fe(III) reducers. Two organisms, Geothermobacter ehrlichii and archaeal strain 140, produced copious amounts of an exopolysaccharide material, which may have played an active role in the dissolution of the structural iron in SWa-1 smectite. The reduction of structural Fe(III) in SWa-1 by archaeal strain 140 was studied in detail. Microbial Fe(III) reduction was accompanied by an increase in interlayer and octahedral charges and some incorporation of potassium and magnesium into the smectite structure. However, these changes in the major element chemistry of SWa-1 smectite did not result in the formation of an illite-like structure, as reported for a mesophilic Fe(III) reducer. These results suggest that thermophilic Fe(III)-reducing organisms differ in their ability to reduce and solubilize structural Fe(III) in SWa-1 smectite and that SWa-1 is not easily transformed to illite by these organisms.
During this first year, we also investigated the dissimilatory reduction of poorly crystalline Fe(III) oxide, and U(VI) reduction at 100C by Pyrobaculum islandicum, in order to gain insight into the significance of biotic metal reduction and mineral formation in hyperthermophilic environments. When P. islandicum was grown in a medium with poorly crystalline Fe(III) oxide as an electron acceptor and hydrogen as an electron donor, the Fe(III) oxide was reduced to an extracellular, ultrafine-grained magnetite with characteristics similar to that found in some hot environments and that was previously thought to be of abiotic origin. Furthermore, cell suspensions of P. islandicum rapidly reduced the soluble and oxidized form of uranium, U(VI), to extracellular precipitates of the highly insoluble U(IV) mineral, uraninite (UO2). The reduction of U(VI) was dependent upon the presence of hydrogen as the electron donor. These findings suggest that microbes may play a key role in metal
deposition in hyperthermophilic environments and provide a plausible explanation for such phenomena as magnetite accumulation and formation of uranium deposits at ca. 100C.
PARTICIPANTS: Evgenya Shelobolina (collaborator) Bruce M. Moskowitz (collaborator) W. C. Elliott (collaborator)
Impacts
Nothing to report at this time.
Publications
- Kashefi, K., B. M. Moskowitz, and D. R. Lovley. (2008). Localization and characterization of magnetite and uraninite production during dissimilatory iron- and uranium-reduction by Pyrobaculum islandicum. Geobiology (in press).
- Kashefi, K., E. S. Shelobolina, W. C. Elliott, and D. R. Lovley. (2008). Growth of Thermophilic and Hyperthermophilic Fe(III)-Reducing Microorganisms on a Ferruginous Smectite as the Sole Electron Acceptor. Appl. Environ. Microbiol. 74 (1): 251-258.
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